Bit by Bit, the Structure of the Potassium Ion Channel Emerges

Image: Courtesy of Roderick MacKinnon MAPPING THE PATH: The transmembrane pore of K+ channels is composed of four identical subunits, of which two are shown. The ion pathway contains a narrow selectivity filter (yellow) and a wide central cavity (asterisk). Three helical elements include the outer helix (M1), pore helix (P), and inner helix (M2). The gate is formed by the inner helix Bundle. (Reprinted with permission from Nature © 2002) Underlying every thought, heartbeat and movement are ion channels, busily conducting high-speed streams of ions into cells, like subway trains coursing through tunnels. Ion channels span the cellular membrane, forming passageways, or pores, through which ions flow down electrochemical gradients. Despite extensive studies of ion channels dating back more than 50 years, crucial structural pieces of the puzzle were still missing that would explain how the high-throughput transmission of ions is achieved, how channels discriminate between ions, and how they open and close in a fraction of a millisecond. One team is helping put those pieces together. Since 1998, Roderick MacKinnon, head of molecular neurobiology and biophysics, Rockefeller University, and Howard Hughes Medical Institute (HHMI) investigator, and colleagues have published papers revealing the potassium and chloride channel structures.1,2 In May the group published its latest findings, which show what the potassium channel looks like when it is open and how the calcium-mediated gating mechanism works like an on-off switch, controlling potassium ion flow through the channel.3,4 THE HISTORY CHAPTER Ion channels are found in every living thing. There are channels for potassium, sodium, calcium, and chloride ions, which may be regulated by voltage, cyclic nucleotides, or neurotransmitters. Their hallmark features include discriminating selectivity, rapid-fire conduction rates, and gating. In 1952, British scientists A.L. Hodgkin and A.F. Huxley first described the flow of potassium and sodium ions in the squid giant axon in response to changes in membrane action potential, garnering them a Nobel Prize in 1963.5 Studying potassium flux, Hodgkin, along with Richard Keynes, later proposed that potassium ions cross membranes through tubes or along negative charges.6 During the 1960s and 1970s, Bertil Hille and Clay Armstrong each independently proposed that these conductors were holes, or channels that had conduction, selection, and gating functions.7 But questions in need of answers still remained: What do the channels look like; how do they conduct ions; and how do metabolic cues regulate channel gating. In 1998, Mac-Kinnon's team published the first crystal structure of the potassium channel, known as the KcsA channel, from the bacterium Streptomyces lividans.1 Since both prokaryotic and eukaryotic pores are closely related, the channel's architectural design is essentially the same for higher organisms as well. "In nature, there are many kinds of potassium channels, but they have one thing in common--the part that makes the ion pathway for ions to pass through," says MacKinnon. "It's very similar for all potassium channels." In each subunit, a helix faces the pore while an outer helix faces the lipid membrane. Four inner helices converge together in a bundle, much like an inverted teepee, on the cell membrane's intracellular side. Armstrong, now a professor of physiology at the University of Pennsylvania, describes the channel as looking like an inverted ice cream cone, where the ice cream is the selectivity filter, which he and others first proposed to exist nearly 30 years earlier. Bonnie Wallace at the Department of Crystallography, University of London, says that MacKinnon's 1998 paper was "the most important new information in structure-function. Full stop. It's that important. In that single, simple structure, it explained the wealth of physiological and pharmacological data over the past 20 years." MacKinnon's work shows what the channel ultimately looks like, as well as the chemistry underlying how potassium ions flow through the channel. Furthermore, adds Hille, now professor in the Department of Physiology and Biophysics, University of Washington, Seattle, MacKinnon's structures confirms the utility and accuracy of the techniques that have been used in the field for years. PICKING THE RIGHT ION But simply knowing what channels look like is just the beginning. It is important to understand why they are so good at selecting potassium over sodium--two ions that are so similar and yet relatively structureless. These channels exhibit a 10,000-fold greater selection of potassium over sodium ions. Without such selectivity, living things would die. And this is where the selectivity filter fits in. Photo: Courtesy of Roderick MacKinnon Roderick MacKinnon Last fall, MacKinnon's group showed the KscA channel at a crisp resolution of 2Å.8,9 Christopher Miller of Brandeis University, Waltham, Mass., and HHMI investigator, wrote the accompanying commentary.10 These papers illustrate the mechanism of potassium hydration and dehydration, ion conduction, and the role of the selectivity filter. To pass through the channel, potassium ions leave their water-filled environment and enter the selectivity filter, which is lined with oxygen atoms from surrounding carbonyl groups. These oxygen atoms mimic a water-filled environment. At this point, the channel discriminates, determining which ions are allowed to pass. The oxygen atoms cradle the potassium ions as snugly as the water did. The cradle is a bit too large for the smaller sodium ions, which remain behind in the water environment where they are more stable. Thus, the channel achieves both specificity and a thermodynamically favorable environment conducive to potassium ion flow. "The potassium ions move into the inner vestibule of the pore, retaining their hydration," explains Armstrong. "To get into the selectivity filter, they lose their hydration, so the ions can't tell if they're surrounded by the oxygen of the [surrounding] carbonyl groups or those of the water." NEW DISCOVERIES The most recent papers from the MacKinnon group reveal the open-channel conformation and the calcium-mediated gating mechanism of the potassium channel in the archeon bacterium, Methanobacterium thermoautotrophicum.3,4 Unable to open the KscA channel for X-ray crystallography studies, the team used the MthK channel instead, which has the same overall structure. The open conformation of the pore, they showed, was surprisingly large. They found a conserved glycine residue, located deep within the membrane, that functions as the gating hinge and permits the large conformational rearrangement. "The open-state structure is really beautiful. It's the sort of thing you look at and say, this really makes sense," comments Rick Aldrich, professor of molecular and cellular physiology, Stanford University, and HHMI investigator. "Having a structure like this sort of immediately gives information on the possible ways to pharmacologically modulate the function." MacKinnon's group also described the gating ring of the potassium channel that permits ion entry.4 Energy from calcium binding triggers conformational changes at the ring's flexible part, pulling open the inner helices so that potassium ions can flow through. However, John Adelman, Vollum Institute, Oregon Health and Sciences University, Portland, OR, is not certain that the gating-ring mechanism that MacKinnon describes explains the entire gating story for the cyclic nucleotide-gated channels.11 "There's a body of literature that postulates [that] the selectivity filter also acts as a gate for [these] channels," he notes. Steven Siegelbaum, Department of Pharmacology, Columbia University, and HHMI investigator, agrees, based on findings from his lab and others: "Different channels may employ slightly different mechanisms for gating. You can't completely rule out significant changes taking place elsewhere." MacKinnon also concurs, adding that the structure of the cyclic nucleotide-gated channel will shed light on the gating mechanism once it is known. Nevertheless, adds Adelman, "[MacKinnon] really has shown us how the process of ion conduction happens and that it's a conserved process throughout nature." Earlier this year, MacKinnon's group also reported the first structure of the chloride channel.2,12 "The chloride channel is a very different situation," says Brandeis' Miller. Miller first proposed that the chloride channel was constructed like a double-barrell shotgun. MacKinnon's structure shows that Miller, his former postdoc supervisor, was right. Furthermore, it reveals for the first time the chemistry underlying chloride selectivity, with chloride ions binding at the membrane's center. "The structure resolved the controversy and opened a whole lot of questions," explains Miller. Compared to the potassium channel, the chloride channel has a very complex and confusing topology. "As for trying to figure out the physiology, the structure is not going to tell us very much," says Miller. MacKinnon agrees, "Only a combination of structural and functional work will lead us to an understanding of how the chloride channel works." MacKinnon says his current focus is on the voltage-dependent potassium channel as well as the chloride channel's gating mechanism. To MacKinnon, the physcian-turned-electrophysiologist-turned-crystallographer, "the fun really begins once you have the structure." Nicole Johnston (johnstnj@mcmaster.ca) is a freelance writer in Hamilton, Ontario, Canada. References 1. D.A. Doyle et al., "The structure of the potassium channel: molecular basis of K+ conduction and selectivity," Science, 280:69-77, 1998. (Cited in more than 1200 papers) 2. R. Dutzler et al., "X-ray structure of a ClC chloride channel at 3.0 Å reveals the molecular basis of anion selectivity," Nature, 415:287-94, Jan. 17, 2002. 3. Y. Jiang et al., "The open pore conformation of potassium channels," Nature, 417:523-6, May 30, 2002. 4. Y. Jiang et al., "Crystal structure and mechanism of a calcium-gated potassium channel," Nature, 417:515-22, May 30, 2002. 5. A.L. Hodgkin, A.F. Huxley, "Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo," Journal of Physiology- London, 116:449-72, 1952. 6. A.L. Hodgkin, R.D. Keynes, "The potassium permeability of a giant nerve fibre," Journal of Physiology-London, 117:500-44, 1952. 7. B. Hille et al., "Ion channels: from idea to reality," Nature Medicine, 5:1105-9, 1999. 8. Y. Zhou et al., "Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 Å resolution," Nature, 414:43-8, Nov. 1, 2001. 9. J.H. Morais-Cabral et al., "Energetic optimization of ion conduction rate by the K+ selectivity filter," Nature, 414:37-42, Nov. 1, 2001. 10. C. Miller, "See potassium run," Nature, 414:23-4, Nov. 1, 2001. 11. M. Schumacher et al., "An open and shut case," Nature, 417:501-2, May 30, 2002. 12. T.J. Jentsch, "Chloride channels are different," Nature, 415:276-7, Jan. 17, 2002.

Bit by Bit, the Structure of the Potassium Ion Channel Emerges

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Image: Courtesy of Roderick MacKinnon
MAPPING THE PATH: The transmembrane pore of K⁺ channels is composed of four identical subunits, of which two are shown. The ion pathway contains a narrow selectivity filter (yellow) and a wide central cavity (asterisk). Three helical elements include the outer helix (M1), pore helix (P), and inner helix (M2). The gate is formed by the inner helix Bundle. (Reprinted with permission from Nature © 2002)